The isolation and purification of nucleic acids (DNA and RNA, for example) from complex matrices such as blood, bacterial cell culture media, and forensic samples is an important process in genetic research, nucleic acid probe diagnostics, forensic DNA testing and other areas. The separation of single-stranded from double-stranded DNA, and of bound from unbound nucleic acid hybridization probes are also important techniques in these areas. A variety of methods for preparing nucleic acids are known in the art; however, each has its limitations.
Traditionally a phenol chloroform extraction has been used, but this requires the use of toxic and corrosive chemicals and is not easily automated. Solid phase extraction has also been used for nucleic acid purification. For example, Boom et al. (U.S. Pat. No. 5,234,809) describe a method for isolating nucleic acids from a nucleic acid source in which a suspension of silica particles is mixed with a buffered chaotropic agent such as guanidinium thiocyanate in a reaction vessel followed by addition of the sample and thorough mixing. In the presence of the chaotrope, the nucleic acids are adsorbed onto the silica, which is separated from the liquid phase by centrifugation, washed with an alcohol water mix, and finally eluted using a dilute aqueous buffer. Silica solid phase extraction requires the use of the alcohol wash step to remove residual chaotrope without eluting the nucleic acid; however, great care must be taken to remove all traces of the alcohol (by heat evaporation or washing with another very volatile and flammable solvent) in order to prevent inhibition of sensitive enzymes used to amplify or modify the nucleic acid in subsequent steps.
Ion exchange methods, such as those offered by Qiagen (Valencia, Calif. 91355), produce high quality nucleic acids. However, these result in the presence of high levels of salts which must be removed before the nucleic acids can be further utilized.
The present invention provides methods for the isolation, including concentration, and preferably purification and recovery of nucleic acids. It also provides methods for the reduction in the amount of nucleic acid that adheres to a surface. In one embodiment, the method involves adhering nucleic acid to a hydrophobic organic polymeric material, such as polypropylene powder and polytetrafluoroethylene fibrils, and removing (e.g., eluting) the nucleic acids from such hydrophobic materials with a nonionic surfactant. In another embodiment, a nonionic surfactant is used to treat a hydrophobic surface to reduce, and preferably prevent, the adhesion of nucleic acids to hydrophobic surfaces.
Nucleic acids isolated according to the invention, will be useful, for example, in assays for detection of the presence of a particular nucleic acid in a sample. Such assays are important in the prediction and diagnosis of disease, forensic medicine, epidemiology, and public health. For example, isolated DNA may be subjected to hybridization and/or amplification to detect the presence of an infectious virus or a mutant gene in an individual, allowing determination of the probability that the individual will suffer from a disease of infectious or genetic origin. The ability to detect an infectious virus or a mutation in one sample among the hundreds or thousands of samples being screened takes on substantial importance in the early diagnosis or epidemiology of an at-risk population for disease, e.g., the early detection of HIV infection, cancer or susceptibility to cancer, or in the screening of newborns for diseases, where early detection may be instrumental in diagnosis and treatment. In addition, the method can also be used in basic research laboratories to isolate nucleic acid from cultured cells or biochemical reactions. The nucleic acid can be used for enzymatic modification such as restriction enzyme digestion, sequencing, and amplification.
In one preferred embodiment, a method for isolating nucleic acid from a sample includes: introducing a sample comprising target nucleic acid (e.g., DNA, RNA, PNA) to a hydrophobic organic polymeric solid phase to adhere at least a portion of the target nucleic acid to the solid phase; and applying a nonionic surfactant to the solid phase to remove at least a portion of the adhered target nucleic acid. Preferably, the sample is a biological sample, which include, for example cells. In certain embodiments, prior to introducing the biological sample, the method includes lysing the cells to release the contents of the cells as a lysate that includes nucleic acid.
In certain embodiments, the method further includes introducing a binding buffer comprising an added salt to the hydrophobic organic polymeric solid phase to assist in adhering the nucleic acid to the solid phase. Preferably, the binding buffer is introduced prior to introducing the sample. In certain embodiments, the method further includes washing the solid phase having nucleic acid adhered thereto to remove non-nucleic acid components of the sample. Such washing is typically with a washing buffer, comprising an added salt.
The hydrophobic organic polymeric solid phase material preferably includes a fluorinated polymer such as polytetrafluoroethylene. More preferably it includes a polyolefin such as polyethylene or polypropylene. The nonionic surfactant is preferably a polyoxyethylene surfactant and more preferably a polyoxyethylene-co-oxypropylene surfactant.
The present invention also provides a method for isolating double-stranded DNA from a sample. The method includes: introducing a sample comprising double-stranded DNA to a hydrophobic organic polymeric solid phase to adhere at least a portion of the double stranded DNA to the solid phase; washing the solid phase having double-stranded DNA adhered thereto to remove non-double-stranded DNA components of the sample (including single-stranded DNA); and applying a nonionic surfactant to the solid phase to remove at least a portion of the adhered double-stranded DNA. Thus, the methods of the present invention include the separation of various types of nucleic acid.
The present invention also provides a method for reducing the amount of (and preferably preventing) nucleic acid that adheres to a hydrophobic organic polymeric surface. The method includes applying a nonionic surfactant to the hydrophobic organic polymeric surface, washing the surface with a solvent (such as water or other solvent, such as that in which the surfactant is dissolved), and contacting the surface with a sample comprising the nucleic acid.
The present invention also provides a kit that includes a hydrophobic organic polymeric solid phase to which nucleic acid will adhere and a nonionic surfactant capable of removing at least a portion of the nucleic acid from the solid phase. Preferably, the kit further includes a flow-through receptacle.
Definitions
xe2x80x9cNucleic acidxe2x80x9d shall have the meaning known in the art and refers to both DNA and RNA, in a wide variety of forms, including, without limitation, double-stranded or single-stranded configurations, circular form, plasmids, relatively short oligonucleotides, peptide nucleic acids also called PNA""s (as described in Nielsen et al., Chem. Soc. Rev., 26, 73-78 (1997)), and the like.
xe2x80x9cIsolatedxe2x80x9d refers to nucleic acid that has been removed from the sample in which it is originally found. This includes simply concentrating the desired nucleic acid without necessarily removing any other materials other than the original solvent in the original sample. It also includes separating desired nucleic acid from other materials, e.g., cellular components such as proteins, lipids, salts, etc. More preferably, the isolated nucleic acid is substantially purified. xe2x80x9cSubstantially purifiedxe2x80x9d refers to nucleic acid that is at least 50%, preferably at least 80%, and more preferably at least 95%, pure with respect to removal of a contaminant, e.g., cellular components such as protein, lipid, or salt. These percentages refer to the amount of target nucleic acid (e.g., DNA, RNA, PNA) relative to the total amount of the target nucleic acid plus other (non-target) nucleic acid and contaminants, e.g., cellular components such as proteins, lipids, salts, etc., other than the solvent in the sample. Thus, the term xe2x80x9csubstantially purifiedxe2x80x9d generally refers to separation of a majority of cellular components or reaction contaminants from the sample, so that compounds capable of interfering with the subsequent use of the isolated nucleic acid are removed.
xe2x80x9cAdheres toxe2x80x9d or xe2x80x9cadherancexe2x80x9d or xe2x80x9cbindingxe2x80x9d refers to reversible binding via a wide variety of mechanisms, including weak forces such as Van der Waals interactions, electrostatic interactions, affinity binding, or physical trapping. The use of this term does not imply a mechanism of action, and includes adsorptive and absorptive mechanisms.
xe2x80x9cHydrophobic organic polymeric solid phasexe2x80x9d refers to a polymer made of repeating units, which may be the same or different, of organic compounds of natural and/or synthetic origin. This includes homopolymers and heteropolymers (e.g., copolymers, terpolymers, tetrapolymers, etc., which may be random or block, for example). A hydrophobic polymer has a critical surface tension of less than the surface tension of water (e.g., less than about 72 dynes/cm), and preferably less than the critical surface tension of nylon e.g., less than about 43 dynes/cm). This term includes fibrous or particulate forms of a polymer, which can be readily prepared by methods well-known in the art. Such materials typically form a porous matrix, although for certain embodiments, the solid phase also refers to a solid surface, such as a nonporous sheet of organic polymeric material.
xe2x80x9cSurfactantxe2x80x9d refers to a substance that lowers the surface or interfacial tension of the medium in which it is dissolved. xe2x80x9cNonionic surfactantxe2x80x9d refers to a surfactant molecule whose polar group is not electrically charged.